CN105176904A - Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof - Google Patents
Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof Download PDFInfo
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- CN105176904A CN105176904A CN201510664099.XA CN201510664099A CN105176904A CN 105176904 A CN105176904 A CN 105176904A CN 201510664099 A CN201510664099 A CN 201510664099A CN 105176904 A CN105176904 A CN 105176904A
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- 238000010353 genetic engineering Methods 0.000 title abstract description 5
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- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 claims abstract description 40
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides genetic engineering strain Streptomyces tsukubaensis L21. The fungus preservation unit is CGMCC (China General Microbiological Culture Collection Center), the preservation date is August 19, 2015, and the preservation number is CGMCC No. 11253. According to the genetic engineering strain Streptomyces tsukubaensis L21, the strain genetic character and the fermentation unit are stable, culture and fermentation conditions are suitable for industrial production, and the fermentation unit is high. The copy number of specific controlling genes in the Tacrolimus creature biosynthetic pathway is increased through genetic engineering, and the genes improve rate-limiting enzyme in the Tacrolimus creature biosynthetic pathway so as to increase the Tacrolimus yield, reduce the cost in the industrial production process and improves the Tacrolimus fermentation unit to 945 mg/L.
Description
Technical field
The invention belongs to bioengineering field, relate to the engineering strain streptomyces tsukubaensis L21 in field of microbial pharmacy and cultural method thereof.
Background technology
Tacrolimus (Tacrolimus) has another name called FK506, be from streptomyces (
streptomyces) in isolated tunning, its chemical structure belongs to 23 membered macrolide microbiotic.There is very strong immunosuppressive activity and anti-mycotic activity, be mainly used for the immunological rejection after treating organs transplanting and treatment skin inflammation etc. clinically.As a kind of neotype immunosuppressant of brute force, mainly through suppressing the release of interleukin-2 (L-2), suppress the lymphocytic effect of T, comparatively ciclosporin (CsA) is strong 100 times comprehensively.In recent years, as a line medication of liver, renal transplantation, in listing such as country such as 14, Japan, the U.S. etc.Clinical experiment shows, its application in the heart, lung, intestines, marrow etc. are transplanted has good curative effect.FK506 also plays positive effect in the autoimmune disorders such as treatment atopic dermatitis (AD), systemic lupus erythematous (SLE), Autoimmune ophthalmopathy simultaneously.
Find that producing the streptomycete of tacrolimus has a lot now, some inherited character and fermentation unit more stable, Culture and fermentation conditions is suitable for suitability for industrialized production, but the fermentation unit of bacterial strain is lower.
Summary of the invention
The object of this invention is to provide a kind of engineering strain streptomyces tsukubaensis L21, described bacterium called after streptomyces tsukubaensis L21(
streptomycestsukubaensisl21), compared with Chinese invention patent (CN200810019003.4) Streptomyces YN06-1593, tacrolimus output increased 72%.This bacterium depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC), preservation day: on August 19th, 2015, preserving number: CGMCCNo.11253, preservation address: No. 3, No. 1, North Star West Road, Chaoyang City, Beijing institute.
Described engineering strain streptomyces tsukubaensis L21 has the nucleotide sequence of the SEQIDNO1 compared with high copy number, this nucleotide sequence coded biosynthetic transcriptional regulator of tacrolimus, most important to the biosynthesizing of tacrolimus.In engineering strain streptomyces tsukubaensis L21, the encoding gene of this regulatory factor is carried out high expression level.
Another object of the present invention is to provide the application of described streptomyces tsukubaensis L21 in tacrolimus biosynthesizing.This bacterial strain continuous several times goes down to posterity at slant medium, fermenting experiment checking is carried out through seed culture medium and fermention medium, by the output of tacrolimus in high-performance liquid chromatogram determination mycelia and fermented liquid, and compare with Chinese invention patent (CN200810019003.4) Streptomyces YN06-1593.The stabilization characteristics of genetics of result display L21, tacrolimus fermentation unit can reach 945mg/L.
A kind of engineering strain streptomyces tsukubaensis L21 provided by the invention, is cultivated by following steps and obtains tacrolimus:
(1) solid culture: streptomyces tsukubaensis L21 is inoculated in ISP4 solid medium, cultivates 7 days in 28 DEG C of incubators;
(2) liquid culture: after solid culture, is inoculated in mycelium in seed culture medium, and 24h cultivated by 28 DEG C of shaking tables, 250rpm, be seeded to after seed culture medium 24h in fermention medium, inoculum size 10%, cultivate 96h post analysis 28 DEG C of shaking table top fermentations and detect tacrolimus.
Described ISP4 solid medium is: starch 1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.2%, ammonium sulfate 0.1%, agar 2%, and described percentage composition is mass percentage.
Described seed culture medium is: starch 2%, dextrin 1%, meat peptone 3%, calcium carbonate 0.2%, and all the other are water, pH6.8, and described percentage composition is mass percentage.
Described fermention medium is: dextrin 4%, meat peptone 1%, corn steep liquor 2%, dipotassium hydrogen phosphate 0.2%, calcium carbonate 0.2%, and all the other are water, pH6.8, and described percentage composition is mass percentage.
The present invention utilize information biology and external biochemical reaction to identify screen the transcriptional regulator of tacrolimus biosynthetic pathway in the streptomyces tsukubaensis obtained, the oligogene that this regulatory factor can activate on tacrolimus biological synthesis gene cluster is expressed, thus strengthens the biosynthesizing of tacrolimus.The expression amount being improved this transcriptional modulatory gene by genetic engineering technique obtains a series of mutant strain, the engineering strain streptomyces tsukubaensis L21 that the fermentation unit filtering out tacrolimus from mutant strain improves.
Bacterial strain inherited character provided by the invention and fermentation unit more stable, Culture and fermentation conditions is suitable for suitability for industrialized production, and fermentation unit is high.The present invention improves the copy number of the specific regulatory control gene in tacrolimus biosynthetic pathway by genetically engineered, as the rate-limiting enzyme in genetic modification tacrolimus biosynthetic pathway to improve the output of tacrolimus, to reduce the cost of commercial process, the fermentation unit of tacrolimus is increased to 945mg/L.The present invention is obtained by the transcriptional regulator of genetic modification tacrolimus biosynthetic pathway, and its tacrolimus output improves 72% than Chinese invention patent (CN200810019003.4) Streptomyces YN06-1593.
Accompanying drawing explanation
Fig. 1 is streptomyces tsukubaensis L21 mycelia and spore scanning electron microscope (SEM) photograph.
Fig. 2 is streptomyces tsukubaensis L21 tacrolimus output during the fermentation.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
The substratum used in embodiment:
1, ISP4 solid medium: starch 1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.2%, ammonium sulfate 0.1%, agar 2%, all the other are water, and described percentage composition is mass percentage.
2, seed culture medium: starch 2%, dextrin 1%, meat peptone 3%, calcium carbonate 0.2%, all the other are water, pH6.8, and described percentage composition is mass percentage.
3, fermention medium: dextrin 4%, meat peptone 1%, corn steep liquor 2%, dipotassium hydrogen phosphate 0.2%, calcium carbonate 0.2%, all the other are water, pH6.8, and described percentage composition is mass percentage.
4, LB substratum: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, all the other are water, and described percentage composition is mass percentage.
5,2 × YT substratum: peptone 1.6%, yeast extract 1%, sodium-chlor 0.5%, all the other are water, and described percentage composition is mass percentage.
embodiment 1the acquisition of streptomyces tsukubaensis L21
Polymerase chain reaction (PCR) is used to increase respectively the DNA sequence dna of SEQIDNO.1, Plastid transformation containing above-mentioned DNA sequence dna by inserting after digestion with restriction enzyme in plasmid pIJ8660, then is contained plasmid pUZ8002 to intestinal bacteria ET12567(by DNA fragmentation after amplification) among.
By the intestinal bacteria ET12567(containing above-mentioned recombinant plasmid containing plasmid pUZ8002) be inoculated in 50mLLB liquid nutrient medium (containing corresponding microbiotic), 37 DEG C are cultured to OD
600be 0.4, collected by centrifugation thalline, washs once with 20mLLB liquid nutrient medium, then washs once with 10mLLB.Then the centrifugal LB liquid nutrient medium abandoning supernatant 0.5mL is resuspended; By 20 μ L screen the streptomyces tsukubaensis spore obtained and add in 2 × YT liquid nutrient medium of 0.5mL resuspended, ice bath cooling 2min after 45 DEG C of heat-shocked 10min; Respectively by being evenly coated on ISP4 solid medium after above-mentioned intestinal bacteria and the mixing of streptomyces tsukubaensis spore, cultivating after 18 hours, adding the Nalidixic Acid of 20 μ g/mL and corresponding microbiotic for 30 DEG C, 30 DEG C are continued cultivation 10 days; Respectively streptomycete transformant obtained above is being collected spore after corresponding antibiotic slant medium cultivates 10 days, merged by PCR method confirmation target dna sequence SEQIDNO.1 and obtain streptomyces tsukubaensis L21 among the genome of screening gained streptomyces tsukubaensis.Described streptomycete called after streptomyces tsukubaensis L21, English name is
streptomycestsukubaensisl21, this bacterium depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation day: on August 19th, 2015, preserving number: CGMCCNO.11253.
embodiment 2the morphological features of streptomyces tsukubaensis L21
Streptomyces tsukubaensis L21 cultivates 7 days at ISP4 solid medium, observe under an electron microscope, can see that the gentle raw hyphal development of substrate mycelium is good, fibrillae of spores is flexible or bending, ripe spore chain spore number is more than 10, and spore is corynebacterium (see figure 1).Appearance features in ISP4 solid medium, the results are shown in Table 1.
embodiment 3the fermentation checking of streptomyces tsukubaensis L21
By 20 μ L streptomyces tsukubaensis L21 spore inoculatings in seed culture medium, rotating speed 250rpm, cultivates 24h for 28 DEG C; Mycelium in seed culture medium is seeded in fermention medium by the inoculum size by 10%, rotating speed 250rpm, cultivates 120 hours for 28 DEG C, distinguishes sampling and measuring tacrolimus output in cultivation after 24,48,72,96,120 hours.
embodiment 4the checking of streptomyces tsukubaensis L21 tacrolimus output
Tacrolimus standard substance methyl alcohol is made into certain density solution, is detected the output of tacrolimus by high performance liquid chromatography, set up tacrolimus concentration---peak area typical curve between the two; Get 1mL fermented liquid, add 1mL methyl alcohol, ultrasonication 30min, centrifuging and taking supernatant, detected the content of tacrolimus by high performance liquid chromatography, obtain the concentration of tacrolimus according to typical curve.Streptomyces tsukubaensis L21 tacrolimus output when fermenting 96h reaches and is 945mg/L to the maximum, the results are shown in Figure 2.
<110> Zhejiang University
<120> engineering strain streptomyces tsukubaensis L21 and application thereof
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Claims (5)
1. a strain gene engineering bacterial strain streptomyces tsukubaensis, is characterized in that: described streptomycete called after streptomyces tsukubaensis L21, English name is
streptomycestsukubaensisl21, this bacterium depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation day: on August 19th, 2015, preserving number: CGMCCNO.11253, preservation address: No. 3, No. 1, North Star West Road, Chaoyang City, Beijing institute.
2. a strain gene engineering bacterial strain streptomyces tsukubaensis according to claim 1, it is characterized in that, there is the nucleotide sequence of the SEQIDNO1 of high copy number, this nucleotide sequence coded biosynthetic transcriptional regulator of tacrolimus, most important to the biosynthesizing of tacrolimus.
3. the application of strain gene engineering bacterial strain streptomyces tsukubaensis L21 in tacrolimus biosynthesizing according to claim 1.
4. application according to claim 3, is characterized in that, is realized by following steps:
(1) solid culture: the inoculation of streptomyces tsukubaensis L21 in ISP4 solid medium, cultivates 7 days in 28 DEG C of incubators;
(2) liquid culture: after solid culture, is inoculated in mycelium in seed culture medium, and 24h cultivated by 28 DEG C of shaking tables, 250rpm, be seeded to after seed culture medium 24h in fermention medium, inoculum size 10%, cultivate 96h post analysis 28 DEG C of shaking table top fermentations and detect tacrolimus.
5. application according to claim 4, is characterized in that: the ISP4 solid medium described in step (1) is: starch 1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.2%, ammonium sulfate 0.1%, agar 2%, all the other are water, and described percentage composition is mass percentage; Seed culture medium described in step (2) is: starch 2%, dextrin 1%, meat peptone 3%, calcium carbonate 0.2%, all the other are water, pH6.8, described percentage composition is mass percentage, and the fermention medium described in step (2) is: dextrin 4%, meat peptone 1%, corn steep liquor 2%, dipotassium hydrogen phosphate 0.2%, calcium carbonate 0.2%, all the other are water, pH6.8, described percentage composition is mass percentage.
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Application publication date: 20151223 Assignee: Zhejiang Qizhen Synthetic Biotechnology Co.,Ltd. Assignor: ZHEJIANG University Contract record no.: X2024980005976 Denomination of invention: Genetic engineering strain Streptomyces Tsukuba L21 and its application Granted publication date: 20190219 License type: Common License Record date: 20240520 |